JP7700092B2 - Glycoform purification - Google Patents

Description

The present invention relates to a method for the separation and purification of glycoforms using ion exchange separation materials having amino acid-based end groups.

Glycans are essential parts of glycoprotein molecules, ensuring their structure and function.
One of the most common glycoproteins is the immunoglobulin, which contains two N-linked oligosaccharides at the conserved asparagine 297 in the CH2 domain of the Fc portion. The structure of the glycan is composed of two N-acetylglucosamine (GlcNAc), three mannose and two GlcNAc residues. Additional monosaccharides such as fucose (Fuc), galactose (Gal), sialic acid containing N-acetylneuraminic acid (NANA) or N-glycolylneuraminic acid (NGNA) residues may also be present. (Figure 1. Glycan structure.) The glycan structure plays an important role in the affinity binding of glycoproteins to receptors. Changes in glycan composition may cause conformational changes of glycoproteins, affecting their specific receptor binding and resulting in changes in effector functions. Moreover, some glycan compositions initiate defensive biological responses, an example being terminal mannoses that bind to effector bearing mannose-binding receptors (ManR).

On the other hand, glycoproteins containing high-terminal mannose glycans, which are commonly found in glycoproteins derived from yeast, insect cells, and plants, may be highly immunogenic in humans (Durocher Y, Butler M. 2009. Expression systems for therapeutic glycoprotein production. Curr Opin Biotechnol 20: 700-707). Therefore, it is crucial to control the levels of high-terminal mannose glycans in biopharmaceutical glycoproteins to avoid potential immunogenicity.

Furthermore, terminal mannose and hybrid glycan structures reduce the conformational stability of monoclonal antibody (mAb) CH2 domains. This may lead to higher levels of enzymatic degradation or shorter storage times for such molecules (Fang, J. Richardson J, Du Z, Zhang Z. Effect of Fc-Glycan Structure on the Conformational Stability of IgG Revealed by Hydrogen/Deuterium Exchange and Limited Proteolysis, Biochemistry 2016, 55, 860-868).

Hybrid glycosylation variants are often formed in the Golgi apparatus. Hybrid glycosylation variants exhibit reduced or altered glycosylation, in other words, do not have the desired glycosylation pattern. Some examples of hybrid forms include variants lacking N-acetylglucosamine in the G0 variant (e.g., G0-N) or galactose in the G1 variant (e.g., G1-N) (Costa AR, Rodrigues ME, Henriques M, Oliveira R, Azeredo J. Glycosylation: impact, control and improvement during therapeutic protein production, Crit Rev Biotechnol. 2013, 1-19). Both mentioned hybrid variants have terminal mannose, thereby increasing the probability of a shorter life span in blood (Goetze AM, Liu YD, Zhang Z, Shah B, Lee E, Bondarenko PV, Flynn GC. 2011. High mannose glycans on the Fc region of therapeutic IgG antibodies increase serum clearance in humans. Glycobiology 21: 949-959).

Moreover, lower levels of galactose affect the activity of glycoproteins, reducing complement-dependent cytotoxicity (CDC) activity. Studies have shown that there may be a 2-fold decrease in activity between mAbs containing G2-glycoforms and those containing G0-glycoforms (Raju TS. 2008. Terminal sugars of Fc glycans influence antibody effector functions of IgGs. Curr Opin Immunol 20: 471-478). Therefore, the ability to control or reduce the amount of hybrid glycosylation variants, especially those with terminal mannose or lacking galactose, would increase the longevity and efficacy of glycoproteins.

One of the most obvious effects of glycan structure changes is seen by the presence or absence of fucose. Fucose is added to glycan structures in the Golgi apparatus. It is known that the presence of fucose in the core glycan structure of a mAb inhibits its binding to the FcγRIIIa receptor, thereby reducing antibody-dependent cell-mediated cytotoxicity (ADCC) activity. Specific binding to the FcγRIIIa receptor can be reduced by 50-fold, thereby having a profound effect on the efficacy of the glycoprotein (Peipp et al, Antibody fucosylation differentially impacts cytotoxicity mediated by NK and PMN effector cells, BLOOD, September 15, 2008, Vol. 112, No. 6, 2390-2399).

It is also well known that the absence of glycosylation dramatically reduces the binding affinity between glycoproteins and receptors. For example, the lack of glycosylation on a mAb dramatically reduces binding to the FcγRI receptor and abolishes binding to the FcγRII and FcγRIII receptors (Liu L, Antibody Glycosylation and Its Impact on the Pharmacokinetics and Pharmacodynamics of Monoclonal Antibodies and FC-Fusion Proteins, Journal of Pharmaceutical Sciences 104: 1866-1884, 2015).

The US Food and Drug Administration (FDA) has assessed glycosylation similarity as one of the most important requirements for biosimilar drugs (FDA, 2012. Guidance for industry quality considerations in demonstrating biosimilarity to a reference protein product). Moreover, improved glycosylation is one of the main trends of biobetters.

For all these reasons, there is a growing need in industry to improve the efficiency of glycosylated biopharmaceutical molecules by controlling, enriching or isolating the various glycan species to enable optimal pharmacokinetics, efficacy, half-life and tolerability.

The current state of the art for glycoprotein separation based on its glycan variants can be performed in a preparative chromatography mode using ion exchange chromatography to enrich for high mannose glycoforms (WO2014/100117). Unfortunately, the enrichment of high mannose-containing glycoforms in the given example overlaps with the enrichment of aggregates, which makes it difficult to recognize, if at all, separation of aggregate-free glycoproteins is also achieved. Moreover, there is no indication that this technique can be used for other glycan variant separation or removal.

An alternative technique for high mannose-containing glycoforms is affinity chromatography using lectins (US20020164328). Although this technique is more specific like ion exchange chromatography, it is limited to high mannose-containing glycans and requires specific binding conditions. In addition, leaching of lectins, regeneration and lifetime of the resin prevent its application to economical high mannose-containing glycan variants.

Other preparative glycan variant separation methods involve a combination of anion exchange and reversed phase chromatography techniques (US20100151584) or the use of anion exchange chromatography alone (IN 01066ch2012). Unfortunately, the prior art lacks techniques to achieve separation of more than one glycoform, addressing the clear need for efficient and effective techniques for separating glycoforms of glycoproteins.

It has been found that ion exchange materials bearing covalently attached leucine or similar residues can be used for glycoform separation and enrichment enabling efficient glycan species separation at capacities of >10 mg glycoprotein/ml material. In more preferred embodiments, the capacity is between 10-80 mg/ml. Moreover, this innovative ion exchange material can be used at high conductivities >10 mS/cm, in more preferred embodiments, the conductivity is between 10-60 mS/cm.

Surprisingly, it was possible to separate and concentrate glycan variants, including high mannose-containing variants, terminal mannose-containing variants, fucose-containing variants and glycosylation-free variants, using a solvent pH gradient. Moreover, this discovery enabled us to explore a broader window of operation in which ionic and hydrophobic interactions contributed to the selectivity of glycan variant separation. In addition, the application of this innovative ion exchange material enabled a broader range of selectivity and improved performance compared to anion exchange mode, showing significant economic advantages compared to affinity chromatography mode.

The present invention is therefore directed to the chromatographic purification and/or separation of protein glycoforms by contacting a sample containing protein glycoforms with a separation material comprising a hydroxyl-containing base matrix having polymer chains covalently grafted to its surface,
a) the base matrix contains hydroxyl groups;
b) the polymer chains are covalently attached to the base matrix via hydroxyl groups;
c) the polymer chain comprises an end group -N(Y)-R3, where
Y are each independently H or CH3, preferably H, and R3 is -CHCOOMR4,
wherein R4 is C1-C4 alkyl, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, preferably isopropyl and isobutyl, very preferably isobutyl, or C1-C4 perfluoroalkyl;
and M is, independently of each other, H, Na, K, or NH4+.

In a preferred embodiment, the method of the present invention comprises the steps of:
a) contacting a sample containing protein glycoforms with a separation material comprising a hydroxyl-containing base matrix having polymer chains covalently attached to its surface, whereby one or more protein glycoforms bind to the separation material;
b) optionally washing the separated material;
c) contacting the separated material with an elution buffer under conditions whereby bound protein glycoforms are eluted from the separated material.
In a preferred embodiment, in step c) the elution buffer has a higher pH than the loading buffer. The elution buffer may be applied as a step or as a gradient.

In a preferred embodiment, in step a), contacting the sample with the separation material is carried out under conditions of increased conductivity, such that the sample loaded onto the separation material has a conductivity between 5 and 60 mS/cm, preferably between 15 and 35 mS/cm. This is typically achieved by adding a salt, such as sodium chloride, to the sample solution.
In another preferred embodiment, the method includes recovering protein glycoforms that flow through the separation material.

In a preferred embodiment, the sample contains mannose rich protein glycoforms.
In another preferred embodiment, the sample contains terminal mannose protein glycoforms.
In another preferred embodiment, the sample comprises fucosylated and nonfucosylated protein glycoforms.

In another preferred embodiment, the sample comprises glycosylated and non-glycosylated proteins.
In another preferred embodiment, the sample comprises antibodies and/or Fc fusion proteins and/or viral protein glycoforms, either isolated or located on the virus or viral capsid.

Preferably, the monomer units of the polymer are linked linearly and each monomer unit comprises an end group --N(Y)--R3.
Preferably, the ionic density of the separation material is between 10 and 1200 μeq/g.
In a preferred embodiment, Y is H and R4 is isopropyl and/or isobutyl.
In a preferred embodiment, the hydroxyl-containing base matrix contains aliphatic hydroxyl groups.

In a preferred embodiment, the base matrix is a copolymer formed by copolymerization of at least one compound from groups a) and b), where a) at least one hydrophilic substituted alkyl vinyl ether represented by formula I
wherein R1, R2, R3 can be each independently H or C1-C6 alkyl, preferably H or _-CH3 ;
and R4 is a radical bearing at least one hydroxyl group, and b) at least one crosslinker according to formula II and/or III and/or IV, where
in which X is a divalent alkyl radical having 2 to 5 C atoms, preferably 2 or 3 C atoms, in which one or more methylene groups which are not adjacent and are not located in the immediate vicinity of N may be replaced by O, C═O, S, S═O, SO ₂ , NH, NOH or N, and one or more H atoms of the methylene groups may be replaced, independently of one another, by hydroxyl groups, C1-C6-alkyl, halogen, NH ₂ , C5-C10-aryl, NH-(C1-C8)-alkyl, N-(C1-C8) _-alkyl2, C1-C6-alkoxy or C1-C6-alkyl-OH, and
In the formulae III and IV, Y1 and Y2 are each independently
C1-C10 alkyl or cycloalkyl, where one or more non-adjacent methylene groups or methylene groups that are not located in the immediate vicinity of N may be replaced by O, C=O, S, S=O, SO2, NH, NOH or N, and one or more H atoms of a methylene group may be replaced, independently of one another, by a hydroxyl group, a C1-C6-alkyl, a halogen, NH2, a _C5 -C10-aryl, an NH-(C1-C8)-alkyl, an N-(C1-C8)-alkyl2 _, a C1-C6-alkoxy or a C1-C6-alkyl-OH,
or C6-C18 aryl, where one or more H in the aryl system may be replaced, independently of one another, by hydroxyl group, C1-C6-alkyl, halogen, _NH2 , NH-(C1-C8)-alkyl, N-(C1-C8) _-alkyl2, C1-C6-alkoxy or C1-C6-alkyl-OH, and
A is a divalent alkyl radical having 2 to 5 C atoms, preferably 2 or 3 C atoms, in which one or more non-adjacent methylene groups or methylene groups that are not located in the immediate vicinity of an N may be replaced by O, C═O, S, S═O, SO ₂ , NH, NOH or N, and one or more H of a methylene group may be replaced, independently of one another, by a hydroxyl group, C1-C6-alkyl, halogen, NH ₂ , C5-C10-aryl, NH(C1-C8)alkyl, N(C1-C8) _alkyl2, C1-C6-alkoxy or C1-C6-alkyl-OH.

R4 in formula I is typically an alkyl radical, a cycloaliphatic radical, or an aryl radical bearing at least one hydroxyl group.
In a highly preferred embodiment, the base matrix is formed by copolymerization of a hydrophilic substituted alkyl vinyl ether selected from the group of 1,4-butanediol monovinyl ether, 1,5-pentanediol monovinyl ether, diethylene glycol monovinyl ether or cyclohexanedimethanol monovinyl ether with divinylethyleneurea (1,3-divinylimidazolin-2-one) as a crosslinker.

In a preferred embodiment, the separation material comprises a hydroxyl-containing base matrix represented by formula V
wherein R1, R2 and Y are each independently H or CH3, preferably H;
R3 is ^-CHCOOMR4 ,
wherein R ⁴ is C1-C4 alkyl, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, preferably isopropyl and isobutyl, very preferably isobutyl, or C1-C4 perfluoroalkyl;
and M is H, Na, K, or _NH4 ;
can be prepared by subjecting monomers to cerium(IV) catalyzed graft polymerization according to

This graft polymerization is preferably carried out according to page 9, Example 8 of US5453186.

In a preferred embodiment, protein glycoforms bind to the separation material at a pH between 2 and 7 and are optionally washed and eluted by increasing the pH value to an alkaline pH, preferably above 9, for example between 9 and 11, preferably around 10.
In a preferred embodiment, the sample is applied to the separation material at an ion density between 10 and 1200 μeq/g.
In a preferred embodiment, between 10 mg and 100 mg of glycoprotein is bound per ml of separated material.

FIG. 1 shows a schematic diagram of glycan structures, where boxes represent N-acetylglucosamine (GlcNAc), fully filled circles—mannose, triangles—fucose (Fuc) and open circles—galactose (Gal). FIG. 2 shows a schematic diagram of a separation material according to the present invention with a substrate (dot) functionalized with a linear polymer constructed by polymerization of acryloylleucine monomers. FIG. 3 shows a schematic diagram of a separation material according to the present invention with a substrate (dot) functionalized with a linear polymer constructed by polymerization of acryloylvaline monomers.

Figure 4 shows the elution peaks of glycoproteins bound on the separation matrix at 20 mg/ml CV load and 250 mM NaCl, the UV adsorption signal trace is the solid line and the pH signal trace is the dashed line. FIG. 5 shows the analytical results of the sample fraction characterization using LC-MS analysis, displayed as the quantitative area of the analyzed elution fractions (1A10-1E3) including 20 mg glycoprotein/ml CV load and loaded glycoprotein (load) at 250 mM NaCl. Figure 6 shows the elution peaks of glycoproteins bound on the separation material at 20 mg/ml CV load and 250 mM NaCl, the UV adsorption signal trace is the solid line and the pH signal trace is the dashed line.

FIG. 7 shows the analytical results of the sample fraction characterization using the LC-MS analytical method displayed as the quantitative area of the analyzed elution fractions (1A10-1E3) including the loaded glycoprotein (load) at 20 mg glycoprotein/ml CV load and 250 mM NaCl. Figure 8 shows the elution peak of glycoprotein (example mAb05) bound on the separation material at 30 mg/ml CV load and 250 mM NaCl. The UV adsorption signal trace is the blue line. FIG. 9 shows the analytical results of the sample fraction characterization using the LC-MS analytical method, displayed as the quantified area of the elution fractions analyzed (1A4-4B2).

Figure 10 shows the elution peak of a glycoprotein (example mAb05) bound onto the separation material at 30 mg/ml CV load and 200 mM NaCl. The UV adsorption signal trace is the solid line and the pH is the dashed line. Figure 11 shows the elution peaks of glycoproteins (as an example, Rituximab®) bound on the separation material at 1 mg/ml CV load and 150 mM NaCl. The UV adsorption signal trace of partially deglycosylated Rituximab® is the solid line, the UV adsorption signal of native Rituximab® is the dotted line, and the pH is the dashed line. Figure 12 shows the flow-through and elution peaks of glycoproteins (example mAb05) bound onto the separation material at 10 mg/ml CV load and 450 mM NaCl. The UV adsorption signal trace is the solid line and the pH is the dashed line.

Figure 13 shows the elution peak of glycoprotein (as an example, spike S1 protein of SARS-CoV-2) bound on the separation material at 1 mg/ml CV load and 150 mM NaCl. The UV adsorption signal trace of glycoprotein is the solid line, and pH is the dashed line. FIG. 14 shows analytical results of sample fraction characterization using HIC analysis, displayed as UV signal traces, including glycoprotein loaded at 1 mg glycoprotein/ml CV load and 250 mM NaCl (load), represented by a solid line, and elution fraction 1C6 of glycoprotein (example spike S1 protein of SARS-CoV-2) represented by a dashed line.

Before describing the invention in detail, it should be understood that this invention is not limited to specific compositions or process steps, as such may vary. It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plurals of the referent unless the context clearly dictates otherwise. Thus, for example, a reference to a "ligand" includes a plurality of ligands, and a reference to an "antibody" includes a plurality of antibodies, etc.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The following terms are defined for purposes of the invention as described herein:

As used herein, the term "target molecule" refers to any molecule, substance or compound that is to be isolated, separated or purified from one or more other components, e.g., impurities, in a sample. An example of a target molecule is a glycoprotein, also called a protein glycoform or a glycan species of a glycoprotein. A target molecule can also be a non-glycosylated protein that is to be separated from a protein glycoform, which is also present in the sample. In the production and/or purification process, the target molecule is typically present in a liquid. The liquid can be water, a buffer, a non-aqueous solvent such as ethanol, or any mixture thereof. Beside the target molecule, the liquid can contain one or more impurities.

The liquid may also be referred to as the sample. The composition of the liquid may vary depending on the process steps performed during production and/or purification. After a chromatographic step, the liquid typically contains a different solvent than before due to the eluent used in the chromatographic step. Typically, only after the very last purification step can the target molecule be dried to prepare the final dosage form.

A glycoprotein or protein glycoform is a glycosylated protein. The glycosylation can be in the form of monosaccharide, disaccharide, or oligosaccharide chains, for example comprising one or more of fucose, mannose, galactose, N-acetylglucosamine, sialic acid, and/or neuraminic acid. For example, antibodies typically have complex type N-linked oligosaccharides. Further information can be found in the introduction above. One example for a common glycan structure is the N-glycoside-linked glycan as found on antibodies.

An "N-glycoside-linked glycan" or "N-glycoside-linked glycan" is typically attached to asparagine 297 (according to Kabat numbering), although complex-type N-glycoside-linked glycans can also be linked to other asparagine residues. Complex-type N-glycoside-linked glycans typically have biantennary complex glycans and primarily have the following structure:
where +/- indicates that a sugar molecule may be present or absent, and the numbers indicate the position of the linkage between the sugar molecules. In the above structure, the sugar chain end attached to the asparagine is called the reducing end (right), and the opposite end is called the non-reducing end. Fucose is usually attached to N-acetylglucosamine ("GIcNAc") at the reducing end, typically by an α1,6 linkage (position 6 of GIcNAc is linked to position 1 of fucose). "Gal" refers to galactose, and "Man" refers to mannose.

Examples of protein glycoforms are proteins with different levels of fucosylation, e.g. different levels of core fucosylation, different levels of sialylation, different levels of galactosylation or different levels of mannosylation. Preferably, the method of the invention is used to separate or purify high mannose protein glycoforms.

High mannose protein glycoforms are glycoproteins that have glycan residues with five or more, typically 5-9, mannose units. One example of a high mannose protein is an antibody with N-linked oligosaccharides containing 5-9 mannose units. Another example of a mannose-rich protein is a viral glycoprotein such as the mannose-rich envelope glycoprotein of HIV1 or the spike (S1) protein of 2019-nCoV (SARS-CoV-2).

A terminal mannose protein glycoform is a glycoprotein having a glycan residue with three mannose units in the core structure, where one or both mannose units in the branch are not linked to N-acetylglucosamine ("GIcNAc"). In some embodiments, this may refer to a G0-N glycoprotein. In some embodiments, this may refer to a G1-N glycoprotein. In some embodiments, this may refer to a hybrid glycoform.

The glycoproteins or protein glycoforms according to the invention can be isolated glycoproteins or glycoproteins linked to other moieties, such as drugs, other proteins, viruses or viral capsids. In some embodiments, the protein glycoforms are produced in mammalian cells, fungal cells, insect cells or plant cells.

The term "antibody" refers to a protein that has the ability to specifically bind to an antigen. "Antibody" or "IgG" further refers to a polypeptide substantially encoded by an immunoglobulin gene or genes, or fragments thereof, that specifically binds to and recognizes an analyte (antigen). Recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta or epsilon, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively.

An exemplary immunoglobulin (antibody) structural unit consists of two pairs of polypeptide chains, each pair having one "light" (about 25 kD) and one "heavy" chain (about 50-70 kD), stabilized, for example, by interchain disulfide bonds. The N-terminus of each chain defines a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains, respectively.

Antibodies may be monoclonal or polyclonal and may exist in monomeric or polymeric form, for example IgM antibodies existing in pentameric form and/or IgA antibodies existing in monomeric, dimeric or multimeric form. Antibodies may also include multispecific antibodies (e.g. bispecific antibodies) and antibody fragments, so long as they retain or are modified to contain a ligand-specific binding domain.

The term "fragment" refers to a portion or part of an antibody or antibody chain that contains fewer amino acid residues than an intact or complete antibody or antibody chain. Fragments can be obtained through chemical or enzymatic treatment of an intact or complete antibody or antibody chain. Fragments can also be obtained by recombinant means. When produced recombinantly, fragments can be expressed alone or as part of a larger protein called a fusion protein. Exemplary fragments include Fab, Fab', F(ab')2, Fc and/or Fv fragments. Exemplary fusion proteins include Fc fusion proteins. In accordance with the present invention, fusion proteins are also encompassed by the term "antibody".

In some embodiments, the antibody is an Fc region-containing protein, e.g., an immunoglobulin. In some embodiments, the Fc region-containing protein is a recombinant protein that includes the Fc region of an immunoglobulin fused to another polypeptide or fragment thereof.

Exemplary polypeptides include, by way of example, renin; growth hormones, including human growth hormone and bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoproteins; alpha-1-antitrypsin; insulin alpha chain; insulin beta chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; clotting factors, such as factor VIIIC, factor IX, tissue factor, and von Willebrand factor; anticoagulants, such as protein C; atrial natriuretic factor; pulmonary surfactant; plasminogen activators, such as urokinase or tissue-type plasminogen activator (t-PA); bombesin; thrombin; hematopoietic growth factors; tumor necrosis factor-alpha and -beta; enkephalinase; RANTES (regulated on activation normally T-cell expressed and secreted); human macrophage inflammatory protein (MIP-1-α); serum albumins such as human serum albumin; Müllerian inhibitory substance; relaxin α chain; relaxin β chain; prorelaxin; mouse gonadotropin-related peptide; microbial proteins such as β-lactamase; DNase; IgE; cytotoxic T-lymphocyte-associated antigen (CTLA) (e.g. CTLA-4); inhibin; activin; vascular endothelial growth factor (VEGF); hormone receptors for neurotransmitters or growth factors; protein A or D; rheumatoid factors; neurotrophic factors such as bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or nerve growth factors such as NGF-β; platelet-derived growth factor (PDGF); fibroblast growth factors such as αFGF and βFGF; epidermal growth factor (EGF); TGF-alpha, and TGF-β1, TGF-β2, TGF-β3, TGF-β4, TGF-β5, TGF-β6, TGF-β7, TGF-β8, TGF-β9, TGF-β10, TGF-β11, TGF-β12, TGF-β13, TGF-β14, TGF-β15, TGF-β16, TGF-β17, TGF-β18, TGF-β19, TGF-β210, TGF-β22, TGF-β310, TGF-β19, TGF-β23, TGF-β19, TGF-β24, TGF-β19, TGF-β25, TGF-β19, TGF-β26, TGF-β19, TGF-β27, TGF-β19, TGF-β28, TGF-β29, TGF-β310, TGF-β111, TGF-β12, TGF-β29, TGF-β19, TGF-β29, TGF-β19, TGF-β29, TGF-β29, TGF-β310, TGF-β1 ... Transforming growth factors (TGFs) such as TGF-β, including TGF-β3, TGF-β4, or TGF-β5; insulin-like growth factors-I and -II (IGF-I and IGF-II); des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor binding proteins (IGFBPs); CD proteins such as CD3, CD4, CD8, CD19, CD20, CD34, and CD40; erythropoietin; bone morphogenetic factors; immunotoxins; osteogenic proteins. protein (BMP); interferons such as interferon-α, -β, and -γ; colony stimulating factors (CSF), such as M-CSF, GM-CSF, and G-CSF; interleukins (IL), such as IL-I to IL-IO; superoxide dismutase; T cell receptors; surface membrane proteins; decay accelerating factors; viral antigens, such as portions of the AIDS envelope; transport proteins; homing receptors; addressins; regulatory proteins; integrins, such as CD1 Ia, CD1 Ib, CD1 Ic, CD 18, ICAM, VLA-4, and VCAM; tumor associated antigens, such as HER2, HER3, or HER4 receptors; and fragments and/or variants of any of the above listed polypeptides.

In addition, an antibody according to the present invention is any protein or polypeptide, fragment or variant thereof, that specifically binds to any of the polypeptides listed above.

As used herein, and unless otherwise stated, the term "sample" refers to any composition or mixture containing a target molecule. A sample may be derived from a biological or other source. Biological sources include eukaryotic sources, such as animals or humans. A preferred sample is a blood or plasma sample derived from a mammal. A sample may also include diluents, buffers, detergents, contaminating species, and the like, that are found mixed with the target molecule. A sample may be "partially purified" (i.e., subjected to one or more purification steps, such as filtration or centrifugation steps) or may be obtained directly from an organism that produces the target molecule. A plasma sample is any sample that contains plasma or a portion of plasma.

The term "impurity" or "contaminant" as used herein refers to any foreign or undesirable molecule, including biological macromolecules such as DNA, RNA, one or more host cell proteins, nucleic acids, endotoxins, lipids, impurities of synthetic origin, and one or more additives, that may be present in a sample containing a target molecule to be separated from one or more foreign or undesirable molecules. The term "impurity" or "contaminant" as used herein can also apply to certain immunoglobulins that need to be separated from the target molecule, such as immunoglobulin A and immunoglobulin M, which cause allergic reactions in patients. In addition, such impurities may include any reagents used in the steps of the production and/or purification process.

The terms "purify," "separate," or "isolate," as used interchangeably herein, refer to increasing the purity of a target molecule by separating it from a composition or sample that contains the target molecule and one or more other components, e.g., impurities. Typically, the purity of a target molecule is increased by removing (completely or partially) at least one impurity from the composition.

The term "chromatography" refers to any type of technique that separates an analyte of interest (e.g., a target molecule) from other molecules present in a mixture. Most often, the target molecule is separated from other molecules as a result of differences in the rate at which individual molecules of the mixture move through a stationary medium or separation material under the influence of a mobile phase or in a bind-elute process. Examples of chromatographic separation processes are reversed-phase chromatography, ion-exchange chromatography, size-exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, and mixed-mode chromatography.

A "buffer" is a solution that resists changes in pH due to the action of its acid-base conjugate components. For example, various buffers that can be used depending on the desired pH of the buffer are described in Buffers. A Guide for the Preparation and Use of Buffers in Biological Systems, Gueffroy, D., ed. Calbiochem Corporation (1975). Non-limiting examples of buffers include MES, MOPS, MOPSO, Tris, HEPES, phosphate, acetate, citrate, succinate, glycine and ammonium buffers, and combinations thereof.

The terms "separation material" or "chromatography matrix" are used interchangeably herein and refer to any type of particulate adsorbent, resin, matrix or solid phase that separates a target molecule (e.g., an Fc region-containing protein such as an immunoglobulin) from other molecules present in a mixture during a separation process. Most often, the target molecule is separated from other molecules as a result of differences in the rate at which individual molecules of the mixture move through and interact with the separation material under the influence of a mobile phase or in a bind-elute process. Separation materials, consisting of resin particles, membranes or monolithic adsorbents by way of example, can be contained in columns or cartridges. Typically, separation materials include a base matrix as a substrate and one or more types of ligands attached to the base matrix.

A "ligand" is or contains a functional group that is attached to a chromatographic base matrix and determines or influences the binding properties of the matrix. Preferably, the ligand is a polymer chain bearing one or more, preferably multiple, functional groups. Most preferred are ligands made of polymer chains bearing at least one functional group per monomer unit that builds them.

Examples of "ligands" include, but are not limited to, ion exchange groups, hydrophobic interaction groups, hydrophilic interaction groups, thiophilic interaction groups, metal affinity groups, affinity groups, bioaffinity groups, and mixed mode groups (combinations of the foregoing). Preferred ligands that may be used herein include, but are not limited to, weak ion exchange groups such as carboxylic acids.

The terms "ion exchange" and "ion exchange chromatography" refer to a chromatographic process in which a target molecule (e.g., an Fc region-containing target protein) in a mixture interacts with charged compounds linked (such as by covalent attachment) to an ion exchange matrix, whereby the target molecule interacts nonspecifically with compounds that are more or less charged than solute impurities or contaminants in the mixture. Impurities in the mixture elute faster or slower than the target molecule from a column of ion exchange material, or are bound to or removed from the resin relative to the target molecule.

"Ion exchange chromatography" specifically includes cation exchange, anion exchange, and mixed-mode ion exchange chromatography. Ion exchange chromatography can either bind to the target molecule (e.g., an Fc region-containing target protein) followed by elution, or can primarily bind to impurities while the target molecule "flows through" the column. Preferably, the ion exchange chromatography step is performed in bind-elute mode.

The phrase "ion exchange matrix" refers to a chromatography medium or separation material that is negatively charged (i.e., a cation exchange resin) or positively charged (i.e., an anion exchange resin). The charge may be provided by attaching one or more charged ligands to the matrix, for example by covalent linkage. Alternatively, or in addition, the charge may be an inherent property of the matrix.
The term "cation exchange matrix" is used herein to refer to a separation material having one or more negatively charged ligands, such as carboxylic acid groups, attached thereto.

When "loading" a separation column in bind-elute mode, a buffer is used to load a sample or composition containing a target molecule (e.g., an Fc region-containing target protein) and one or more impurities onto a chromatography column (e.g., an ion exchange column). The buffer has a conductivity and/or pH such that the target molecule binds to the separation material while ideally all impurities do not bind to the column and flow through the column.

When "loading" a separation column to allow the target molecule to "flow through," a buffer is used to load a sample or composition containing the target molecule (e.g., an Fc region-containing target protein) and one or more impurities onto a chromatography column (e.g., an ion exchange column). The buffer has a conductivity and/or pH such that the target molecule flows through the column without binding to the separation material, while ideally all impurities bind to the column.

When "loading" a separation column to "bind and elute" a target molecule, a buffer is used to load a sample or composition containing a target molecule (e.g., an Fc region-containing target protein) and one or more impurities onto a chromatography column (e.g., an ion exchange column). The buffer has a conductivity and/or pH such that the target molecule binds to the separation material. Separation from the one or more impurities occurs subsequently with a conductivity and/or pH such that the target molecule is washed or eluted before or after the one or more impurities. Typically, the buffer in which the sample is loaded onto the separation material is called a loading buffer or sample buffer.

The term "equilibration" refers to the use of a buffer to equilibrate the separation material prior to loading the target molecules. Typically, a loading buffer is used for equilibration.
"Washing" or "washing" a separation material means passing a suitable liquid, e.g., a buffer, through or over the separation material. Typically, washing is used after loading to remove weakly bound contaminants from the separation material before eluting the target molecules and/or to remove unbound or weakly bound target molecules.

In this case, typically the wash buffer and the loading buffer are the same. If a virus inactivation buffer is used, it is used to inactivate the given viruses present before eluting the target molecule. In this case, typically the virus inactivation buffer is different from the loading buffer because it may contain detergent/detergents or have different properties (pH/conductivity/salts and their amounts).

Washing can also be used to remove contaminants from the separation material after elution of the target molecules. This is done by passing a suitable liquid, e.g. a buffer, through or over the separation material after elution of the target molecules. In this case, the washing buffer is typically different from the loading buffer. It may contain detergent/detergents or have different properties (pH/conductivity/salts and their amounts). The washing buffer is for example an acidic buffer.

"Eluting" a molecule (e.g., a polypeptide of interest such as immunoglobulin G or an impurity) from a separation material means removing the molecule therefrom. Elution can be performed directly in flow-through mode, when the target molecule is eluted with a solvent in front of the loading buffer, or by changing the solution conditions so that a buffer different from the loading buffer competes with the molecule of interest for the ligand sites on the separation material. A non-limiting example is eluting a molecule from an ion exchange material by changing the ionic strength of the buffer surrounding the ion exchange material so that the buffer competes with the molecule for the charged sites of the ion exchange material.

The term "mean particle size" or d50 means the average particle size distribution value at 50% of the cumulative particle size distribution. Particle size is determined by laser diffraction, preferably using a Malvern's Master Sizer.
The term "average pore size" means the average pore size distribution value at 50% of the cumulative pore size distribution.
The terms "flow-through process,""flow-throughmode," and "flow-through operation," as used interchangeably herein, refer to a separation technique in which at least one target molecule (e.g., an Fc region-containing protein or an antibody) contained in a sample along with one or more impurities is intended to flow through a separation material that typically binds one or more impurities, while the target molecule typically is unbound and elutes (i.e., flows through) from the separation material along with the loading buffer.

The terms "bind and elute mode" and "bind and elute process" as used herein refer to a separation technique in which at least one target molecule (e.g., an Fc region-containing protein) contained in a sample binds to a suitable separation material (e.g., an ion exchange chromatography medium) and is then eluted with a buffer different from the loading buffer.

The term "ion density" as used herein refers to the number of ions per unit of volume or mass of a given separation material, more specifically, the number of ions of a given type (e.g., cations or anions) per unit volume or mass of the separation material. Most often, the number of ions is estimated by titrating a given separation material. Moreover, the amount of ions is given in equivalents (eq) per mass or unit volume of the separation material.

The term "conductivity" as used herein refers to an intrinsic property of most materials that quantifies how strongly it resists or conducts electric current. In aqueous solutions, such as buffers, electric current is carried by charged ions. Conductivity is determined by the number of charged ions, the amount of charge they carry, and how fast they move. Thus, for most aqueous solutions, the higher the concentration of dissolved salts, the higher the conductivity. Increasing the temperature allows the ions to move faster, thus increasing the conductivity. Typically, unless otherwise indicated, conductivity is defined at room temperature. The base unit of conductivity is the Siemens (S). It is defined as the reciprocal of the resistance, expressed in ohms, measured between the opposing faces of a 1 cm cube of liquid. Values are therefore estimated in S/cm.

The present invention provides a method for separating or concentrating protein glycoforms. This means that one or more protein glycoforms can be separated from other protein glycoforms and/or from other impurities in a sample. Preferably, at least one protein glycoform is separated from at least one other glycoform, for example one high mannose glycoform is separated from other protein glycoforms. This is done by chromatographic separation on a given separation material, also called a resin, which comprises a base matrix to which polymer chains are double bond-attached. The polymer chains are made of amino acids and monomers that contain polymerizable double bonds.

The method of the invention allows glycoforms to be separated, enriched and/or purified, making efficient glycan species separation feasible. In certain aspects of the invention, it is useful for separating glycan variants containing high mannose or terminal mannose molecules, which are more hydrophobic and demonstrate faster clearance. Moreover, such glycan variants may contribute to higher toxicity and lower efficacy of glycoproteins. In another aspect of the invention, it is useful for separating glycoproteins that retain fucose, which ensures better control of antibody-dependent cell-mediated cytotoxicity (ADCC) activity. In other aspects of the invention, it is useful for removing proteins that are not glycosylated but are otherwise identical to the target glycoprotein.

In one embodiment of the invention, the purified glycoprotein does not contain high mannose glycan variants. In another embodiment of the invention, the purified glycoprotein contains low levels of hybrid glycan variants with terminal mannose. In another embodiment of the invention, the purified glycoprotein contains low levels of non-fucosylated glycan variants. In another embodiment of the invention, the purified glycoprotein contains low levels of non-glycosylated proteins.

The separation material of the present invention comprises a substrate having attached, preferably grafted, tentacle-like structures.

The substrate, also called base matrix, contains reactive groups available for the graft polymerization reaction, in particular OH groups, preferably aliphatic OH groups. The substrate can therefore also be prepared, for example, from organic polymers.

Organic polymers of this type can be polysaccharides such as agarose, dextran, starch, cellulose, etc., or synthetic polymers such as poly(acrylamides), poly(methacrylamides), poly(acrylates), poly(methacrylates), hydrophilically substituted poly(alkylaryl ethers), hydrophilically substituted poly(alkylvinyl ethers), poly(vinyl alcohols), poly(styrene) and copolymers of the corresponding monomers. These organic polymers can also be preferably employed in the form of crosslinked hydrophilic networks. This also includes polymers made from styrene and divinylbenzene, which, like other hydrophobic polymers, can preferably be employed in hydrophilized form.

Alternatively, inorganic materials such as silica, zirconium oxide, titanium dioxide, aluminum oxide, etc. may be employed as substrates. It is equally possible to employ composite materials, i.e. particles which themselves can be magnetized, for example by copolymerization of magnetizable particles or magnetizable cores. It is also possible to use core-shell materials, where the shell, i.e. at least the surface or coating, bears OH groups.

However, since the materials according to the invention should preferably withstand alkaline cleaning or regeneration, e.g. at basic pH, over extended periods of use, preference is given to the use of hydrophilic substrates which are hydrolytically stable or cannot be hydrolyzed without difficulty.

The base matrix may consist of irregularly shaped or spherical particles, the particle size of which may be between 2 and 1000 μm. Preference is given to an average particle size between 3 and 300 μm, and in the most preferred embodiment the average particle size is between 20 and 63 μm.
The base matrix may in particular be in the form of non-porous or preferably porous particles. The average pore size may be between 2 and 300 nm. Preference is given to pore sizes between 5 and 200 nm, and the most preferred average pore size is between 40 and 110 nm.

The base matrix may equally well be in the form of a membrane, a fiber, a hollow fiber, a coating or a monolithic molding. The monolithic molding is preferably in the form of a porous three-dimensional body, for example a cylinder.

In a preferred embodiment, the base matrix is a copolymer formed by copolymerization of at least one compound from groups a) and b), where a) at least one hydrophilic substituted alkyl vinyl ether represented by formula I
wherein R1, R2, R3 can be, independently of each other, H or C1-C6 alkyl, preferably H or -CH3;
and R4 is a radical bearing at least one hydroxyl group, and b) at least one crosslinker according to formula II and/or III and/or IV, where
in which X is a divalent alkyl radical having 2 to 5 C atoms, preferably 2 or 3 C atoms, in which one or more methylene groups which are not adjacent and are not located in the immediate vicinity of N may be replaced by O, C═O, S, S═O, SO ₂ , NH, NOH or N, and one or more H atoms of the methylene groups may be replaced, independently of one another, by hydroxyl groups, C1-C6-alkyl, halogen, NH ₂ , C5-C10-aryl, NH-(C1-C8)-alkyl, N-(C1-C8) _-alkyl2, C1-C6-alkoxy or C1-C6-alkyl-OH, and
In the formulae III and IV, Y1 and Y2 are each independently
C1-C10 alkyl or cycloalkyl, where one or more non-adjacent methylene groups or methylene groups that are not located in the immediate vicinity of N may be replaced by O, C=O, S, S=O, SO2, NH, NOH or N, and one or more H atoms of a methylene group may be replaced, independently of one another, by a hydroxyl group, a C1-C6-alkyl, a halogen, NH2, a _C5 -C10-aryl, an NH-(C1-C8)-alkyl, an N-(C1-C8)-alkyl2 _, a C1-C6-alkoxy or a C1-C6-alkyl-OH,
or C6-C18 aryl, where one or more H in the aryl system may be replaced, independently of one another, by hydroxyl group, C1-C6-alkyl, halogen, _NH2 , NH-(C1-C8)-alkyl, N-(C1-C8) _-alkyl2, C1-C6-alkoxy or C1-C6-alkyl-OH, and
A is a divalent alkyl radical having 2 to 5 C atoms, preferably 2 or 3 C atoms, in which one or more non-adjacent methylene groups or methylene groups that are not located in the immediate vicinity of an N may be replaced by O, C═O, S, S═O, SO ₂ , NH, NOH or N, and one or more H of a methylene group may be replaced, independently of one another, by a hydroxyl group, C1-C6-alkyl, halogen, NH ₂ , C5-C10-aryl, NH(C1-C8)alkyl, N(C1-C8) _alkyl2, C1-C6-alkoxy or C1-C6-alkyl-OH.

R4 in formula I is typically an alkyl radical, a cycloaliphatic radical, or an aryl radical bearing at least one hydroxyl group.

In a highly preferred embodiment, the base matrix is formed by copolymerization of a hydrophilic substituted alkyl vinyl ether taken from the group of 1,4-butanediol monovinyl ether, 1,5-pentanediol monovinyl ether, diethylene glycol monovinyl ether or cyclohexanedimethanol monovinyl ether with divinylethyleneurea (1,3-divinylimidazolin-2-one) as a crosslinker.
An example of a suitable commercially available vinyl ether-based substrate is Eshmuno®, Merck KGaA, Germany.

wherein linear polymer chains are covalently attached to a surface of a substrate such that a separation material is produced;
a) the substrate contains hydroxyl groups, preferably aliphatic;
b) the polymer is covalently attached to the support;
c) the polymer contains amino acid residues;
d) The monomer units of the polymer are linked in a linear fashion.

The actual separation material, which comprises a substrate and doubly attached linear polymer chains, can be prepared in a variety of ways. In the "grafting onto" method, the polymer chains must first be formed from monomers and are attached to the surface in a second step. In the "grafting from" method, the polymerization reaction is initiated on the surface and the graft polymer is built up directly from the individual monomers. Other polymerization methods that allow attachment of the substrate to the surface can also be employed.

Preference is given to the "grafting from" method, and especially to variants in which only small amounts of by-products, such as non-covalently bound polymers, are formed, which must be removed by separation. Processes involving controlled free radical polymerization, such as for example the method of atom transfer free radical polymerization (ATRP), appear to be of particular interest. Here, initiator groups are covalently bound to the support surface with the desired density in a first step. The initiator groups are, for example, halides bound via ester functions, as in 2-bromo-2-methylpropionic acid ester. The graft polymerization is carried out in a second step in the presence of copper(I) salts.

A highly preferred one-step graft polymerization reaction suitable for the production of the separation materials used in the present invention can be initiated by cerium(IV) on a hydroxyl-containing substrate without the need to activate the substrate.

The cerium(IV)-initiated grafting is preferably carried out according to EP 0 337 144 or US 5,453,186. The chains produced are linked to the substrate via monomer units. For this purpose, the substrate according to the invention is suspended in a solution of monomers, preferably in an aqueous solution. The grafting-on of the polymeric material can be carried out in the course of a conventional redox polymerization with the exclusion of oxygen. The polymerization catalyst employed is the cerium(IV) ion, since this catalyst forms free radical sites on the surface of the substrate, from which the polymerization of the monomers is initiated. The reaction is usually carried out in dilute mineral acid. To carry out the graft polymerization, the acid is usually employed in aqueous solution, with a concentration in the range of 1 to 0.00001 mol/l, preferably 0.1 to 0.001 mol/l. Particular preference is given to the use of dilute nitric acid, employed in a concentration of 0.1 to 0.001 mol/l.

For the preparation of separation materials according to the invention, the monomers are usually added in excess relative to the substrate. Typically, 0.05 to 100 mol of total monomer per liter of deposited polymeric material is employed, preferably 0.05 to 25 mol/l.

The polymerization is terminated by a termination reaction involving the cerium salt. For this reason, the (average) chain length can be influenced by the concentration ratio of substrate, initiator and monomer. Furthermore, homogeneous or even mixtures of different monomers can be employed; in the latter case, graft copolymers are formed.

The monomers conveniently used for the preparation of the separation materials used in the present invention are represented by the formula V
This is in accordance with
wherein R ¹ , R ² and Y are each independently H or CH ₃ , preferably H.
^R3 is ^-CHCOOMR4 ,
wherein R ⁴ is C1-C4 alkyl, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, preferably isopropyl and isobutyl, very preferably isobutyl, or C1-C4 perfluoroalkyl;
and M is H, Na, K, or _NH4 ⁺ .

Perfluoroalkyl means that all H atoms in the alkyl residue are replaced with F atoms.
Exemplary preferred structures of formula V are
In the formula, R ¹ , R ² and Y are H;
^R3 is ^-CHCOOMR4 ,
wherein R ⁴ is isopropyl;
and M is H;
And formula Vb
In the formula, R ¹ , R ² and Y are H;
^R3 is ^-CHCOOMR4 ,
In the formula, ^R4 is isobutyl;
and M is H;
It is.

Such monomers may also be described as acryloyl valine (formula Va), acryloyl leucine (formula Vb), acryloyl alanine, acryloyl norleucine, methacryloyl valine, methacryloyl leucine, methacryloyl alanine, methacryloyl norleucine, dimethacryloyl valine, dimethacryloyl leucine, dimethacryloyl alanine, dimethacryloyl norleucine, with acryloyl valine and acryloyl leucine being preferred, and acryloyl leucine being especially preferred.

The separation materials used in the method of the present invention preferably contain only tentacle-like linear polymer structures grafted onto a substrate constructed from monomers according to formula V. Preferably, they contain linear polymers constructed only by one type of monomer according to formula V.

However, it is also possible that a linear polymer is constructed by copolymerization of two or more different monomers according to formula V. It is also possible that a linear polymer is constructed by copolymerization of one or more different monomers according to formula V and one or more other polymerizable monomers, such as other acrylamides, methacrylates, acrylates, methacrylates functionalized (e.g., with ionic, hydrophilic or hydrophobic groups), etc.

Exemplary structures of separation materials according to the present invention are shown in Figures 2 and 3. Figure 2 shows a substrate (dots) functionalized with a linear polymer constructed by polymerization of acryloyl leucine monomers. Additionally, for clarity, the carboxyalkyl end groups of each polymer unit are shown. Figure 3 shows a substrate (dots) functionalized with a linear polymer constructed by polymerization of acryloyl valine monomers. Additionally, for clarity, the carboxyalkyl end groups of each polymer unit are shown.

In a preferred embodiment, the separation material used in the method of the present invention is an ion exchange material of various amounts of ion density bands ranging from 10 to 1200 μeq/g, where in a more preferred embodiment the ion density bands are between 400 and 900 μeq/g.
In a preferred embodiment, the ion exchange material can contain an ion exchange functional group and a hydrophobic functional group, where in a more preferred embodiment both functional groups are on a single surface functional unit resulting from one monomeric unit built into a polymer chain, where in a most preferred embodiment the functional group is part of an amino acid residue.

Typically, chromatography columns comprising the separation material described above are used in the method according to the invention. Chromatography columns are known to those skilled in the art. They typically comprise a cylindrical tube or cartridge filled with the separation material, as well as a filter and/or a means for fixing the separation material in the tube or cartridge, and a connection for solvent delivery into and out of the tube. The size of the chromatography column varies depending on the application, e.g. analytical or preparative. The chromatography column can also be a membrane containing cartridge.

The materials used in the method of the invention can also be described as substrates provided with separation effectors. They can be used for selective, partially selective or non-selective binding or adsorption of one or more target components, aiming at their separation out of the sample liquid, or for selective, partially selective or non-selective binding or adsorption of one or more secondary components, aiming at the separation of secondary components out of the matrix, isolation, enrichment and/or depletion of biopolymers from natural sources, isolation, enrichment and/or depletion of biopolymers from recombinant sources, isolation, enrichment and/or depletion of proteins or polypeptides, isolation, enrichment and/or depletion of monoclonal and polyclonal antibodies, isolation, enrichment and/or depletion of viruses, isolation, enrichment and/or depletion of host cell proteins or isolation, enrichment and/or depletion of glycoproteins. Preferred target molecules are glycoproteins.

The target molecule is separated from one or more other substances from a sample, in which the sample containing the target molecule is dissolved in a liquid, which is brought into contact with the material according to the invention. The contact time is usually in the range of 30 seconds to 24 hours. It is advantageous to work according to the principle of liquid chromatography by passing the liquid through a chromatography column containing the separation material according to the invention. The liquid can pass through the column simply through its gravity or by being pumped through it by means of a pump.

An alternative method is batch chromatography, in which the target molecules or biopolymers are mixed with the liquid by stirring or shaking for the length of time required to allow them to bind to the separation material. Similarly, it is possible to work according to the principle of a chromatographic fluidized bed by introducing the liquid to be separated into, for example, a suspension containing the separation material, where the separation material is selected to be suitable for the desired separation due to its high density and/or magnetic core.

If the chromatographic process is operated in bind-elute mode, the target molecules bind to the separation material. The separation material can then be washed with a wash buffer, which preferably has the same ionic strength and the same pH as the liquid in which the target molecules are brought into contact with the separation material. The wash buffer removes all substances that do not bind to the separation material. This may be followed by a further wash step with another suitable buffer, without desorbing the target molecules. Desorption of the bound target molecules is carried out by changing the ionic strength in the eluent and/or by changing the pH in the eluent and/or by changing the solvent. The target molecules are thus obtained in purified and concentrated form in the eluent. The target molecules usually have a purity of 70% to 99%, preferably 85% to 99%, particularly preferably 90% to 99%, after desorption.

It is also possible to use the above bind-elute process to separate or purify more than one target molecule, whereby a group of target molecules binds to the separation material and is separated from one or more impurities as described above.

However, if the chromatographic process is operated in flow-through mode, the target molecule or a group of different types of target molecules remains in the liquid, but other accompanying substances bind to the separation material. The target molecule is then directly obtained by collecting the column eluate in the through-flow. It is within the skill of the art to know how the conditions, especially pH and conductivity, must be adapted to bind a particular biopolymer to the separation material, or whether it is advantageous for the purification task not to bind the target molecule.

The present invention is preferably directed to the use of the above-described separation material for the separation and purification of glycoproteins, as well as to a method for the separation and/or purification of glycoproteins by liquid chromatography, comprising contacting the separation material with a sample containing one or more glycoproteins, preferably under acidic conditions.

Unexpectedly, we have found that the ion exchange material according to the invention can be used for glycoform separation or concentration enabling efficient glycan species separation at capacities of >10 mg glycoprotein/ml material, in more preferred embodiments, capacities between 10-80 mg/ml. Moreover, this innovative ion exchange material can be used at high conductivities >5 mS/cm, in more preferred embodiments, conductivities between 5-60 mS/cm.

Surprisingly, it was possible to separate and concentrate glycan variants, including high mannose-containing variants, terminal mannose-containing variants, fucose-containing variants and glycosylation-free variants, preferably using a pH change from 4-7 to a pH above 9, preferably in gradient or step mode at a pH between 9-11, most preferably up to pH 10. In addition, the application of this separation material showed significant economic advantages compared to affinity chromatography mode and enabled wider selectivity and improved performance compared to anion exchange mode.

Furthermore, the application is not limited to the separation of different glycan variant species, but can also be used to remove non-glycosylated variants from the glycosylated ones.

In addition, the application of this material is not limited to bind-elute applications, but can also be used in flow-through mode, resulting in the adsorption of higher mannose-containing species or higher terminal mannose-containing glycan variants onto the ion-exchange material using buffers with pH<6 and/or high conductivity (>20 mS/cm). Moreover, surprisingly, only this ion-exchange material, which consists primarily of amino acids covalently attached to the material, has the required selectivity for glycan species, in a more preferred embodiment these amino acids being valine or leucine (including combinations and derivatives thereof), most preferably leucine.

Furthermore, the present invention provides a chromatography-based glycoprotein purification step that is regenerative and applicable over a wide operation window, e.g., pH 3-10; conductivity 5-60 mS/cm.
In a preferred embodiment, the ion exchange material used in the present invention is incorporated into a chromatography column and used for the glycoprotein purification process in bind-elute mode to separate glycan species.

In a preferred embodiment, the ion exchange material used in the present invention is incorporated into a chromatography column and used for a glycoprotein purification process in bind-elute mode to separate glycan species, where the operation window spans between pH 2-10, and in a more preferred embodiment the pH is between 4-7.
In a preferred embodiment, the ion exchange material used in the present invention is incorporated into a chromatography column and used for a glycoprotein purification process in bind-elute mode to separate glycan species, where a solvent pH elution is used to recover the bound components.

In a preferred embodiment, the ion exchange material used in the present invention is incorporated into a chromatography column and used for a glycoprotein purification process in bind-elute mode to separate glycan species, where the binding window spans between 10 mg glycoprotein/ml material and 100 mg glycoprotein/ml material, and in a more preferred embodiment the binding window spans between 20 mg glycoprotein/ml material and 80 mg glycoprotein/ml material.

In a preferred embodiment, the ion exchange material used in the present invention is incorporated into a chromatography column and used for a glycoprotein purification process in bind-elute mode to separate glycan species, where the conductivity band span is between 5-60 mS/cm, and in a more preferred embodiment the conductivity range is between 15-35 mS/cm.

In another preferred embodiment, the ion exchange material used in the present invention is incorporated into a chromatography column and used for a glycoprotein purification process in a flow-through mode to separate glycan species, where the low mannose-containing species are in the flow-through and the higher mannose-containing species bind to the separation material.

In another preferred embodiment, the ion exchange material used in the present invention is incorporated into a chromatography column and used for a glycoprotein purification process in flow-through mode to separate glycan species, where non-hybrid species are in the flow-through and hybrid (e.g., terminal mannose-containing) species bind to the separation material.

In another preferred embodiment, the ion exchange material used in the present invention is incorporated into a chromatography column and used for a glycoprotein purification process in a flow-through mode to separate glycan species, where the low fucosylation species are in the flow-through and the high fucosylation-containing species bind to the separation material.

In another preferred embodiment, the method of the present invention comprises the steps of:
a) applying to a chromatography column comprising an ion exchange material used in the present invention a sample comprising a mixture of protein glycoforms, at least one of the glycoforms being a high mannose glycoform;
b) separating and eluting at least one high mannose glycoform in the flow-through;
c) eluting bound glycoforms from the separated material, whereby the eluted glycoforms are depleted in high mannose glycoforms.

In another preferred embodiment, the method of the present invention comprises the steps of:
a) applying a sample comprising a mixture of protein glycoforms, at least one of the glycoforms being a high mannose glycoform, to a chromatography column comprising an ion exchange material used in the present invention, where at least one of the high mannose glycoforms, and typically also other protein glycoforms, bind to the separation material;
b) contacting the separated material with an elution buffer, whereby one or more glycoforms distinct from the high mannose glycoforms are eluted, while at least one high mannose glycoform remains bound to the separated material;
c) optionally contacting the separated material with a second elution buffer, which is typically different from the first elution buffer, and thereby eluting at least one high mannose glycoform.

In this embodiment, the high mannose glycoforms are reduced in the eluate obtained in step b) compared to the sample applied in step a) and enriched in the eluate obtained in step c). The mannose glycoforms separated in the flow-through or binding elution as described above are preferably antibodies with different degrees of mannosylation or viruses and/or viral capsids carrying proteins with different degrees of mannosylation.

In another preferred embodiment, the ion exchange material used in the present invention is incorporated into a chromatography column and used for a glycoprotein purification process in flow-through or in bind-elute mode to separate viruses and/or virus particles containing glycoprotein forms from viruses and/or virus particles containing other types of glycoproteins or not containing glycoproteins. Preferably, the separation occurs based on the different amounts of mannose present in the glycan structures.

In another preferred embodiment, the method of the present invention comprises the steps of:
a) applying to a chromatography column comprising an ion exchange material used in the present invention a sample comprising a mixture of protein glycoforms, at least one of the glycoforms being a terminal mannose glycoform, wherein at least one of the terminal mannose glycoforms, and typically also other protein glycoforms, binds to the separation material;
b) contacting the separated material with an elution buffer, whereby one or more glycoforms different from the terminal mannose glycoform are eluted, while at least one terminal mannose glycoform remains bound to the separated material;
c) optionally contacting the separated material with a second elution buffer, which is typically different from the first elution buffer, and thereby eluting at least one terminal mannose glycoform.

In this embodiment, the terminal mannose glycoforms are reduced in the eluate obtained in step b) compared to the sample applied in step a) and enriched in the eluate obtained in step c). The terminal mannose glycoforms separated in the flow-through or binding elution as described above are preferably antibodies with different degrees of mannosylation or viruses and/or viral capsids carrying proteins with different degrees of mannosylation.

In another preferred embodiment, the ion exchange material used in the present invention is incorporated into a chromatography column and used for a glycoprotein purification process in flow-through or in bind-elute mode to separate viruses and/or virus particles containing glycoprotein forms from viruses and/or virus particles containing other types of glycoproteins or not containing glycoproteins. Preferably, the separation occurs based on the different amounts of terminal mannose present in the glycan structures.

In another preferred embodiment, the method of the present invention comprises the steps of:
a) applying to a chromatography column comprising a separation material used in the present invention a sample comprising a mixture of protein glycoforms, at least one of the glycoforms being a fucose-bearing glycoform, wherein at least one of the fucose-bearing glycoforms, and typically also other protein glycoforms, binds to the separation material;
b) contacting the separated material with an elution buffer, whereby one or more glycoforms distinct from the glycoforms that do not have fucose are eluted, while at least one glycoform that has fucose remains bound to the separated material;
c) optionally contacting the separated material with a second elution buffer, which is typically different from the first elution buffer, and thereby eluting at least one glycoform that does not have fucose.

In this embodiment, the eluate obtained in step b) is depleted of fucose-free glycoforms compared to the sample applied in step a) and is enriched in the eluate obtained in step c). The fucose-containing glycoforms separated in the flow-through or binding elution as described above are preferably antibodies with different degrees of fucosylation or viruses and/or viral capsids carrying proteins with different degrees of fucosylation.

In another preferred embodiment, the ion exchange material used in the present invention is incorporated into a chromatography column and used for a glycoprotein purification process in flow-through or in bind-elute mode to separate viruses and/or virus particles containing glycoprotein forms from viruses and/or virus particles containing other types of glycoproteins or not containing glycoproteins. Preferably, the separation occurs based on the different amounts of fucose present in the glycan structures.

In another preferred embodiment, the method of the present invention comprises the steps of:
a) applying a sample comprising a mixture of glycan-bearing and glycan-free proteins, where at least one of the glycan-bearing proteins, the protein glycoform, binds to a chromatography column comprising the separation material used in the present invention;
b) contacting the separated material with an elution buffer, whereby at least one protein bearing glycans is eluted, while at least one protein without glycans remains bound to the separated material;
c) optionally contacting the separated material with a second elution buffer, typically different from the first elution buffer, and thereby eluting at least one free glycan.

In this embodiment, proteins carrying glycoforms are enriched in the eluate obtained in step b) and depleted in the eluate obtained in step c) compared to the sample applied in step a). The glycoforms separated in the flow-through or binding elution as described above are preferably antibodies with different degrees of glycosylation or viruses and/or viral capsids carrying proteins with different degrees of glycosylation.

In another preferred embodiment, the ion exchange material used in the present invention is incorporated into a chromatography column and used for a glycoprotein purification process in flow-through or in bind-elute mode to separate viruses and/or virus particles containing glycoprotein forms from viruses and/or virus particles containing other types of glycoproteins or not containing glycoproteins. Preferably, the separation occurs based on the different amounts of glycosylation present in the glycan structures.

In another preferred embodiment, the ion exchange material used in the present invention is incorporated into a chromatography column and used for a glycoprotein purification process in a flow-through mode to separate glycan species, where proteins containing glycosylation are in the flow-through and proteins not containing glycosylation bind to the separation material.

In a preferred embodiment, the ion exchange material can be of various particle sizes ranging from 1 to 200 μm, and in a more preferred embodiment, the average particle size is between 20 and 63 μm.
In a preferred embodiment, the ion exchange material according to the present invention may have a variety of pore sizes ranging from 4 to 1500 nm, in a more preferred embodiment the average pore size is between 10 and 120 nm, and in a most preferred embodiment the average pore size is between 40 and 110 nm.

The method of the present invention is very flexible. Any suitable chromatography mode (bind-elute or flow-through) can be applied, and the conditions can be varied within the wide ranges mentioned above. In addition, the target molecule can also be varied. As discussed above, in each case the sample contains at least one protein glycoform. However, the target molecule can be either a protein glycoform, a group of different protein glycoforms, or a non-glycosylated protein.

Typically, in the methods of the invention, at least one protein glycoform binds to the separation material, but this protein glycoform does not necessarily have to be the target molecule. It may be the target molecule, but the target molecule can also be another protein glycoform or a non-glycosylated protein that also binds to the separation material or is in the flow-through. The target molecule can be defined as required, since the process of the invention allows for the separation of different protein glycoforms, and of glycosylated and non-glycosylated proteins.

The present invention is further illustrated by the following figures and examples, without, however, being limited thereto.
The entire disclosures of all applications, patents, and publications cited above and below, as well as in corresponding EP Patent Application No. 19184130.3, filed July 7, 2019, are hereby incorporated by reference.

EXAMPLES The following examples illustrate practical applications of the present invention.

1. Synthesis of isolated materials For the preparation of monomers used for grafting from the process, amino acids, e.g. valine, leucine or alanine, are dissolved in VE water and the pH is adjusted to a pH above 13 by adding NaOH (32%). An acrylic compound, such as acrylic acid chloride or acrylic acid, is added at a temperature between 0-5° C. and the mixture is stirred for 1 hour.
The reaction schemes for valine and leucine with acrylic acid chloride are shown in Figures 2 and 3.

The pH is then adjusted to about 2.2 by adding nitric acid, after which the OH-containing substrate, e.g., Eshmuno® particles, is added.
The polymerization is initiated by adding cerium (IV) nitrate. The reaction is carried out at 30-50° C. for 4 hours.
After the polymerization reaction, non-reacted components and initiators are removed by extensive washing with acidic, basic and solvent mixtures at room or elevated temperatures.

2. Rituximab® and High Mannose Glycan Form Separation Ion exchange material as prepared according to Example 1 (eg, with average particle size between 20-63 μm, average pore size between 40-110 nm and ionic density between 400-900 μeq/g) was evaluated for its ability to separate high mannose-containing species of the marketed drug Rituximab®. The ion exchange material was packed into a chromatographic column of 5×100 mm dimensions with an asymmetry between 0.8-1.2 and >3000 plates/m. After packing the ion exchange material, the resulting chromatographic column was washed with 1 M NaOH solution for 30 min and pre-equilibrated with a loading buffer solution having a pH of 4.75 and 250 mM NaCl. The buffer solution contained a combination of salts such as sodium dihydrogen phosphate, TRIS, and glycine to obtain a pH of 4.5. The same solution was used to dilute the Rituximab® samples down to a concentration of 4.8 mg/ml.

The solution was loaded onto the prepared chromatographic column until a loading of 20 mg Rituximab® per ml resin was reached. These and subsequent steps were performed at a buffer velocity of 150 cm/h. After loading 20 mg/ml Rituximab®, the chromatographic column was washed with a buffer solution of pH 4.75 and then eluted using gradient elution with a buffer having a pH of 8.5 and 250 mM NaCl. The elution buffer was prepared using different salts such as sodium dihydrogen phosphate, TRIS, and glycine. The conductivity and pH values were traced during the experimental setup, which indicates that Rituximab® elution from the column was achieved due to pH changes during gradient elution. The sample eluate was fractionated and the obtained fractions were evaluated for line using LC-MS method for glycan species identification (Figure 4).

Analytical evaluation of the collected fractions using LC-MS analysis is displayed in Figure 5 and shows that the main glycoprotein elution peak, further characterized by representative fractions 1B9 and 1C7, did not contain any high mannose variants. Most high mannose glycan variants were eluted at higher pH, further characterized by representative fractions 1D10 and 1E3 (Figure 5).

As shown in Figure 4, separation/enrichment of high mannose-containing glycan species is possible using linear pH gradient elution. No high mannose variants were detected in the main glycoprotein-containing fractions (e.g., 1B9 and 1C7). All high mannose-containing variants eluted in the separation fractions (e.g., 1D10).

3. Erbitux® and High Mannose Glycan Form Separation in a Wide Application Window in the Range of 200 mM to 600 mM Sodium Sulfate Ion exchange material as prepared according to Example 1 (e.g., with an average particle size between 20-63 μm, an average pore size between 40-110 nm and an ionic density between 400-900 μeq/g) was evaluated for its ability to separate high mannose-containing species of the commercial drug Erbitux®. The ion exchange material was packed into a chromatographic column of 5×100 mm dimensions with an asymmetry between 0.8-1.2 and >3000 plates/m. After packing the ion exchange material, the resulting chromatographic column was washed with 1 M NaOH solution for 30 min and pre-equilibrated with a loading buffer solution having a pH of 4.5 and 250 mM NaCl. The buffer solution contained a combination of salts such as sodium dihydrogen phosphate, TRIS, and glycine to obtain a pH of 4.5.

The pre-purified Erbitux® sample was loaded onto the prepared chromatographic column until a pre-purified Erbitux® load of 5 mg per ml resin was reached. These and subsequent steps were performed at a buffer velocity of 150 cm/h. After loading 5 mg/ml pre-purified Erbitux®, the chromatographic column was washed with a buffer solution of pH 4.5 and various amounts of Na ₂ SO _4. The amount of Na ₂ SO ₄ was varied between 200 and 600 mM. The column was then eluted using gradient elution with a buffer having a pH of 8.5 and the corresponding amount of Na ₂ SO _4. The elution buffer was prepared using different salts such as sodium dihydrogen phosphate, TRIS, and glycine. The conductivity and pH values were traced during the experimental setup, which shows that Erbitux® elution from the column was achieved due to pH changes during gradient elution.

In Table 1, the elution maxima of the main peak and the high mannose-containing peak at the corresponding pH are displayed.

Table 1 shows the elution maxima of the main peak and the high mannose-containing peak at the corresponding pH.

As shown in Table 1, separation/enrichment of high mannose-containing glycan species is possible over a wide range of conductivities using linear pH gradient elution. Fractions with high mannose-containing variants eluted at higher pH values compared to glycan species that do not contain high mannose across the entire range investigated.

4. Rituximab® and Hybrid Glycan Form Separation Ion exchange material as prepared according to Example 1 (eg, with an average particle size between 20-63 μm, an average pore size between 40-110 nm, and an ionic density between 400-900 μeq/g) was evaluated for its ability to separate hybrid glycan species of the marketed drug Rituximab®. The ion exchange material was packed into a chromatographic column of 5×100 mm dimensions with an asymmetry between 0.8-1.2 and >3000 plates/m. After packing the ion exchange material, the resulting chromatographic column was washed with 1 M NaOH solution for 30 min and pre-equilibrated with a loading buffer solution having a pH of 4.75 and 250 mM NaCl. The buffer solution contained a combination of salts such as sodium dihydrogen phosphate, TRIS, and glycine to obtain a pH of 4.75. The same solution was used to dilute the Rituximab® samples down to a concentration of 4.8 mg/ml.

The solution was loaded onto the prepared chromatographic column until a loading of 20 mg Rituximab® per ml resin was reached. These and subsequent steps were performed at a buffer velocity of 150 cm/h. After loading 20 mg/ml Rituximab®, the chromatographic column was washed with a buffer solution of pH 4.75 and then eluted using gradient elution with a buffer having a pH of 8.5 and 250 mM NaCl. The elution buffer was prepared using different salts such as sodium dihydrogen phosphate, TRIS, and glycine. The conductivity and pH values were traced during the experimental setup, which indicates that Rituximab® elution from the column was achieved due to pH changes during gradient elution. The sample eluate was fractionated and the obtained fractions were evaluated for line using LC-MS method for glycan species identification (Figure 7).

Analytical evaluation of the collected fractions using LC-MS analysis is displayed in Figure 7 and shows that the main glycoprotein elution peak, further characterized by representative fractions 1B9 and 1C7, did not contain any hybrid glycan variants (e.g., those containing terminal mannose). Most hybrid glycan variants eluted at higher pH, which were further characterized by representative fractions 1D10 and 1E3 (Figure 7).

As shown in Figures 6 and 7, separation/enrichment of hybrid glycan species is possible using linear pH gradient elution. No hybrid glycan variants were detected in the main glycoprotein-containing fractions (e.g., 1B9 and 1C7). All hybrid glycan variants eluted in the separation fractions (e.g., 1D10 and 1E3).

5. mAb05 and Hybrid Glycan Form Separation Ion exchange material as prepared according to Example 1 (examples: with average particle size between 20-63 μm, average pore size between 40-110 nm and ionic density between 400-900 μeq/g) was evaluated for its ability to separate hybrid glycan species of mAb05. The ion exchange material was packed into a chromatographic column of 5×100 mm dimensions with asymmetry between 0.8-1.2 and >3000 plates/m. After packing the ion exchange material, the resulting chromatographic column was washed with 1 M NaOH solution for 30 min and pre-equilibrated with 250 mM NaCl with a loading buffer solution having a pH of 4.75. The buffer solution contained a combination of salts such as sodium dihydrogen phosphate, TRIS, and glycine to obtain a pH of 4.75. The same solution was used to dilute the mAb05 sample down to a concentration of 5 mg/ml.

The solution was loaded onto the prepared chromatographic column until a loading of 30 mg mAb05 per ml resin was reached. These and subsequent steps were performed at a buffer velocity of 150 cm/h. After loading 30 mg/ml mAb05, the chromatographic column was washed with a buffer solution of pH 4.75 and then eluted using gradient elution with a buffer having a pH of 8.5 and 250 mM NaCl. The elution buffer was prepared using different salts such as sodium dihydrogen phosphate, TRIS, and glycine. The conductivity and pH values were traced during the experimental setup, which indicates that mAb05 elution from the column was achieved due to pH changes during gradient elution. The sample eluate was fractionated and the resulting fractions were evaluated for line using LC-MS method for glycan species identification (Figure 9).

Analytical evaluation of the collected fractions using LC-MS analysis is displayed in Figure 9 and shows that the first elution fractions 1A4-2A4 did not contain any hybrid glycan variants (e.g., terminal mannose-containing G0F-N and G1F-N). Most hybrid glycan variants were eluted at higher pH, which is further characterized by the representative 3B5-4B2 fractions (Figure 9). In addition to the hybrid forms, mannose 5-containing glycan variants were also eluted at higher pH as well.

As shown in Figure 8, separation/enrichment of hybrid glycan species is possible using linear pH gradient elution. No hybrid glycan variants were detected in the main glycoprotein-containing fractions (e.g., 1A4-2A4). All hybrid glycan variants eluted at higher pH values (e.g., 3B5-4B2).

Moreover, the fractions obtained were characterized using analytical size exclusion chromatography and the levels of fragmentation or aggregation were monitored. The results show the presence of less than 1% aggregates in the samples used or in the fractions obtained (Table 2).

Table 2 shows the analytical size exclusion chromatography results for the resulting fractions of the analyzed elution fractions (1B4-1F12) expressed in areas and in percentages for fragmented or aggregated species. All fractions were >99% pure.

6. mAb05 and Fucosylated and Nonfucosylated Forms Separation Ion exchange material as prepared according to Example 1 (eg, with an average particle size between 20-63 μm, an average pore size between 40-110 nm and an ionic density between 400-900 μeq/g) was evaluated for its ability to separate fucosylated and nonfucosylated glycan species of mAb05. The ion exchange material was packed into a chromatographic column of 5×100 mm dimensions with an asymmetry between 0.8-1.2 and >3000 plates/m. After packing the ion exchange material, the resulting chromatographic column was washed with 1 M NaOH solution for 30 min and pre-equilibrated with a loading buffer solution having a pH of 4.75 and 200 mM NaCl. The buffer solution contained a combination of salts such as sodium dihydrogen phosphate, TRIS, and glycine to obtain a pH of 4.75. The same solution was used to dilute the mAb05 sample down to a concentration of 5 mg/ml.

The solution was loaded onto the prepared chromatographic column until a loading of 30 mg mAb05 per ml resin was reached. These and subsequent steps were performed at a buffer velocity of 150 cm/h. After loading 30 mg/ml mAb05, the chromatographic column was washed with a buffer solution of pH 4.75 and then eluted using gradient elution with a buffer having a pH of 8.5 and 200 mM NaCl. The elution buffer was prepared using different salts such as sodium dihydrogen phosphate, TRIS, and glycine. The conductivity and pH values were traced during the experimental setup, which shows that mAb05 elution from the column was achieved due to pH changes during gradient elution (Figure 10). The flow-through and sample eluate were fractionated and the resulting fractions were evaluated for line using LC-MS method for glycan species identification (Table 3).

Analytical evaluation of the collected fractions using LC-MS analysis is displayed in Table 3 and shows that the first elution fractions 1B4-1D5 contained less non-fucosylated forms (G0 and G1) compared to (G0F and G1F) of the loaded glycoprotein. Higher amounts of fucosylated glycan variants (G0F and G1F) were eluted at higher pH values, which is further characterized by representative fractions 1E4 and 1F12 (Table 3). In addition, mannose 5-containing glycan variants were also eluted at higher pH as well.

Table 3 shows the analytical results of the sample fraction characterization using the LC-MS analytical method, expressed as the quantitative area of the analyzed elution fractions (1B4-1F12).

As shown in Figure 10 and Table 3, separation/enrichment of non-fucosylated glycan species is possible using linear pH gradient elution. Less fucosylated glycan variants were detected in the main glycoprotein-containing fractions (e.g., 1B4-1D5). More fucosylated variants eluted at higher pH values (e.g., 1E4-1F12).

7. Separation of native and deglycosylated Rituximab® Ion exchange material as prepared according to Example 1 (eg, with an average particle size between 20-63 μm, an average pore size between 40-110 nm and an ionic density between 400-900 μeq/g) was evaluated for its ability to separate glycosylated and non-glycosylated Rituximab®. The ion exchange material was packed into a chromatographic column of 5×100 mm dimensions with an asymmetry between 0.8-1.2 and >3000 plates/m. After packing the ion exchange material, the resulting chromatographic column was washed with 1 M NaOH solution for 30 min and pre-equilibrated with a loading buffer solution having a pH of 4.75 and 150 mM NaCl. The buffer solution contained a combination of salts such as sodium dihydrogen phosphate, TRIS, and glycine to obtain a pH of 4.75. The same solution was used to dilute native and deglycosylated Rituximab® samples up to a concentration of 4 mg/ml.

The solution was loaded onto the prepared chromatographic column until a loading of 1 mg Rituximab® per ml resin was reached. These and subsequent steps were performed at a buffer velocity of 150 cm/h. After loading 1 mg/ml Rituximab®, the chromatographic column was washed with a buffer solution of pH 4.75 and then eluted using gradient elution with a buffer having a pH of 8.5 and 150 mM NaCl. The elution buffer was prepared using different salts such as sodium dihydrogen phosphate, TRIS, and glycine. The conductivity and pH values were traced during the experimental setup, which indicates that elution of Rituximab® from the column was achieved due to pH changes during gradient elution.

To prepare the samples, Rituximab® was partially deglycosylated using an Endoglycosydase digest.
Partially deglycosylated and native Rituximab® were both loaded in two different applications and the retention times were compared (Figure 11).

The elution profile of a native glycoprotein (e.g., Rituximab®) in a linear pH gradient shows a lower elution pH for the native glycosylated protein. The deglycosylated portion of the glycoprotein elutes at a higher pH in the gradient.

As shown in Figure 11, it is possible to separate/enrich deglycosylated and native glycoproteins using linear pH gradient elution.

8. mAb05 and High Mannose Glycan Form Separation in Flow-Through Mode Ion exchange material as prepared according to Example 1 (examples of mean particle size between 20-63 μm, mean pore size between 40-110 nm and ionic density between 400-900 μeq/g) was evaluated for its ability to separate high mannose glycan species of mAb05 in flow-through mode. The ion exchange material was packed into a chromatographic column of 5×100 mm dimensions with an asymmetry between 0.8-1.2 and >3000 plates/m. After packing the ion exchange material, the resulting chromatographic column was washed with 1 M NaOH solution for 30 min and pre-equilibrated with 400 mM NaCl with a loading buffer solution having a pH of 4.75. The buffer solution contained a combination of salts such as sodium dihydrogen phosphate, TRIS, and glycine to obtain a pH of 4.75. The same solution was used to dilute the mAb05 sample down to a concentration of 4.8 mg/ml.

The solution was loaded onto the prepared chromatographic column until a loading of 10 mg mAb05 per ml resin was reached. These and subsequent steps were performed at a buffer velocity of 150 cm/h. After loading 10 mg/ml mAb05, the chromatographic column was washed with a buffer solution of pH 4.75 and then eluted using gradient elution with a buffer having a pH of 8.5 and 400 mM NaCl. The elution buffer was prepared using different salts such as sodium dihydrogen phosphate, TRIS, and glycine. The conductivity and pH values were traced during the experimental setup, which shows that high mannose mAb05 glycovariants elution from the column was achieved due to pH changes during gradient elution. Meanwhile, glycovariants that do not contain mannose are not bound on the column and are in the flow-through. The flow-through and sample eluate were fractionated and the resulting fractions were evaluated using LC-MS method for glycan species identification (Table 4).

Table 4 shows analytical evaluation of the collected fractions using LC-MS analysis. It shows that the flow-through fraction 1F8 contained <1% mannose-containing glycovariants. Most mannose-containing glycovariants were eluted at higher pH, which is further characterized by the representative fraction 1H9 (Table 4).

As shown in FIG. 12, using linear pH gradient elution, separation/enrichment of low mannose containing glycan species in the flow-through is possible. High mannose glycan variants were detected in the bound fraction (e.g. 1H9). High mannose glycan variants eluted at higher pH values (e.g. 1H9).

9. Separation of SARS-CoV-2 spike S1 protein in bind-elute mode Ion exchange material as prepared according to Example 1 (for example with average particle size between 20-63 μm, average pore size between 40-110 nm and ionic density between 400-900 μeq/g) was evaluated for its ability to bind SARS-CoV-2 spike S1 protein. The ion exchange material was packed into a chromatographic column of 5×100 mm dimensions with an asymmetry between 0.8-1.2 and >3000 plates/m. After packing the ion exchange material, the resulting chromatographic column was washed with 1 M NaOH solution for 30 min and pre-equilibrated with a loading buffer solution having a pH of 4.75 and 250 mM NaCl. The buffer solution contained a combination of salts such as sodium dihydrogen phosphate, TRIS, and glycine to obtain a pH of 4.75. The same solution was used to reconstitute the S1 protein sample to a concentration of 0.6 mg/ml.

The solution was loaded onto the prepared chromatographic column until 1 mg S1 protein loading per ml resin was reached. These and subsequent steps were performed at a buffer velocity of 150 cm/h. After loading 1 mg/ml S1 protein, the chromatographic column was washed with a buffer solution of pH 4.75 and then eluted using gradient elution with a buffer having a pH of 10.5 and 250 mM NaCl. The elution buffer was prepared using different salts such as sodium dihydrogen phosphate, TRIS, and glycine. The conductivity and pH values were traced during the experimental setup, which indicates that S1 protein elution from the column was achieved due to pH changes during gradient elution. The sample eluate was fractionated and the obtained fractions were evaluated for line using analytical HIC method for protein identification (Figure 13).

Analytical evaluation of the collected fractions using analytical HIC method is displayed in Figure 14 and shows that the main elution peak (dashed line), further characterized by the representative 1C6 fraction, contained only S1 protein (solid line). (Figure 14)

As shown in Figure 13, it is possible to separate/concentrate the SARS-CoV-2 spike S1 protein using linear pH gradient elution. The elution peak contains only the S1 protein.

10. mAb03 and High Mannose Glycan Form Separation in Flow-Through Mode Ion exchange material as prepared according to Example 1 (examples of mean particle size between 20-63 μm, mean pore size between 40-110 nm and ionic density between 400-900 μeq/g) was evaluated for its ability to separate high mannose glycan species of mAb03 in flow-through mode. The ion exchange material was packed into a chromatographic column of 5×100 mm dimensions with an asymmetry between 0.8-1.2 and >3000 plates/m. After packing the ion exchange material, the resulting chromatographic column was washed with 1 M NaOH solution for 30 min and pre-equilibrated with 250 mM NaCl with a loading buffer solution having a pH of 5.12. The buffer solution contained a combination of salts such as sodium dihydrogen phosphate, TRIS, and glycine to obtain a pH of 5.12. The same solution was used to dilute the mAb03 sample down to a concentration of 5.0 mg/ml.

The solution was loaded onto the prepared chromatographic column until mAb03 loading per 1 ml resin was reached. These and subsequent steps were performed at a buffer velocity of 150 cm/h. After loading 100 mg/ml mAb03, the chromatographic column was washed with a buffer solution of pH 5.12 and then eluted using gradient elution with a buffer having a pH of 8.5 and 250 mM NaCl. The elution buffer was prepared using different salts such as sodium dihydrogen phosphate, TRIS, and glycine. The conductivity and pH values were traced during the experimental setup, which shows that high mannose mAb03 glycovariants elution from the column was achieved due to pH changes during gradient elution. Meanwhile, glycovariants without mannose are not bound on the column and are in the flow-through. The flow-through was fractionated and the resulting fractions were evaluated for quantification of high mannose species (Table 5).

Table 5 shows analytical assessment of the commulative high mannose species content of collected fractions after a given mAb03 breakthrough. The starting concentration of high mannose species was 7.68%, indicating that the 66.2 mg flow-through mAb03 sample contained <1% high mannose-containing glycovariants.

11. Gammanorm® Static Binding Capacity The static binding capacity of Gammanorm® was measured using materials prepared from hexafluorovaline used for grafting. Hexafluorovaline was dissolved in VE water and the pH was adjusted to above 13 by adding NaOH (32%). An acrylic compound such as acrylic acid chloride or acrylic acid was added at a temperature between 0-5°C and the mixture was stirred for 1 hour. The pH was then adjusted to about 2.2 by adding nitric acid. Afterwards, an OH-containing substrate, for example Eshmuno® particles, was added. The polymerization was started by adding cerium (IV) nitrate. The reaction was carried out at 30-50°C for 4 hours.
Precise amounts of such material were soaked in solutions containing 5 mg/ml Gammanorm at pH 5.0 and varying amounts of NaCl. After 4 hours of incubation, the material was removed and the amount of Gammanorm® remaining in the solution was measured, thereby providing an estimate of the amount of bound Gammanorm®.

The static binding capacities measured were as follows:
At 0 mM NaCl - 64.5 mg Gammanorm®/ml hexafluorovaline grafted material; at 30 mM NaCl - 50.9 mg Gammanorm®/ml hexafluorovaline grafted material; at 75 mM NaCl - 50 mg Gammanorm®/ml hexafluorovaline grafted material bound.

Claims (16)

by contacting a sample containing protein glycoforms with a separation material comprising a base matrix having polymer chains covalently attached to its surface ,
- terminal mannose protein glycoforms,
- fucosylated protein glycoforms,
- non-fucosylated protein glycoforms,
- glycosylated proteins, and
Non-glycosylated proteins
1. A method for the chromatographic purification and/or separation of protein glycoforms, comprising the steps of :
The polymer chain comprises an end group -N(Y)-R3, where
Y are each independently H or _CH3 , and R3 is -CHCOOMR4,
wherein R4 is C1-C4 alkyl or C1-C4 perfluoroalkyl;
and M is H, Na, K, or _NH4 . 2. The method of claim 1, comprising the steps of: a) contacting a separation material with a sample containing protein glycoforms, whereby one or more protein glycoforms bind to the separation material; and b) contacting the separation material with an elution buffer under conditions whereby the bound protein glycoforms are eluted from the separation material. The method of claim 2, further comprising washing the separated material having one or more bound protein glycoforms. The method of any one of claims 1 to 3, characterized in that the method further comprises recovering protein glycoforms that flow through the separated material in step a). The method according to any one of claims 1 to 4, characterized in that the elution buffer has a higher pH than the loading buffer used to contact the sample with the separation material in step a). The method according to any one of claims 1 to 5, characterized in that in step a), the sample brought into contact with the separation material in step a) has a conductivity of 5 to 60 mS/cm. The sample comprises:
The method according to any one of claims 1 to 6, characterized in that the glycoforms comprise one or more of the following: - mannose-rich protein glycoforms; - terminal mannose protein glycoforms; - fucosylated and non-fucosylated protein glycoforms; - glycosylated and non-glycosylated protein glycoforms. The method according to any one of claims 1 to 7, characterized in that the sample contains glycosylated antibodies and/or viral protein glycoforms. The method according to any one of claims 1 to 8, characterized in that the polymer chain is built up of monomer units, the monomer units of the polymer chain are linearly linked, and each monomer unit comprises a terminal group -N(Y)-R3. The method according to any one of claims 1 to 9, characterized in that Y is H and R4 is isopropyl and/or isobutyl. The method according to any one of claims 1 to 10, characterized in that the ion density of the separation material is between 10 and 1200 μeq/g. The method according to any one of claims 1 to 11, characterized in that the protein glycoforms bind to the separation material at a pH between 2 and 7. The method according to any one of claims 1 to 11, characterized in that the protein glycoforms bind to the separation material at a pH between 2 and 7 and are washed and eluted by increasing the pH value to a value between 9 and 11. The method according to any one of claims 1 to 13, characterized in that the sample is applied to the separation material at an ion density between 10 and 1200 μeq/g. The method according to any one of claims 1 to 14, characterized in that between 10 mg and 100 mg of protein glycoforms are bound per ml of separated material. The method according to any one of claims 1 to 15, characterized in that the sample contains SARS-CoV-2 protein glycoforms.